Immunohistochemical localization of bombesin or gastrin releasing peptide (BN/GRP-IR) and substance P in consecutive sections of rat hypothalamus was studied. BN/GRP-IR in the hypothalamus ws characterized by gel filtration column chromatography. The results of this comparative immunohistochemical study show that there are major differences in the distribution and number of neurons exhibiting substance P and BN/GRP-IR in the rat hypothalamus. Cells which exhibit substance P-immunore-activity (IR) are distributed throughout the hypothalamus whereas only two nuclei (nucleus suprachiasmaticus and nucleus paraventricularis) contain large numbers of cells with BN/GRP-IR. The number of substance P-IR cells is also considerably larger than that of BN/GRP-IR cells. The BN/GRP-IR material is not identical to BN but is chromatographically identical to GRP(1-27) and GRP(18-27). The highest concentration of opioid peptide met-enkephalin-arg-gly-leu (YGGFMRGL) was found in the dorsal horn of the rat spinal cord. A dense network of perikarya and fibers was found in laminae I and II of the dorsal horn. Cell bodies were frequently observed in laminae IV. Additional terminals were seen around the central canal and in the ventral grey matter, often outlining perikarya of motor neurons. This localization of YGGFMRGL in spinal cords suggest a role for this peptide in sensory-motor integration.